Regulatory T Cell Expressing a Chimeric Antigen Receptor

Pending Publication Date: 2020-11-12
MILTENYI BIOTEC B V & CO KG +1
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Benefits of technology

[0013]In one embodiment of the invention the CD137 CAR expressed in the Treg is specific for the exogenous soluble antigen dextran. The administration of dextran to a subject in need to be treated with said CAR specific

Problems solved by technology

However, in-vitro Treg populations are in general contaminated with Tc

Method used

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  • Regulatory T Cell Expressing a Chimeric Antigen Receptor
  • Regulatory T Cell Expressing a Chimeric Antigen Receptor
  • Regulatory T Cell Expressing a Chimeric Antigen Receptor

Examples

Experimental program
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Effect test

Example

[0210]Example 1

Generation of Dextran-Specific CAR-Tregs With Different Intracellular Signaling Domains

[0211]The CAR constructs contain a specific binding fragment that is derived from an antibody specific for an exogenous antigen (e.g. dextran). The hinge region may be derived e.g. from IgG domains, CD8a CD8a, or CD28 and may comprise an epitope / tag allowing for the detection of the CAR. The transmembrane domain may be derived e.g. from CD8a or CD28 followed by one to three signaling domains containing CD3ζ and CD137 as for example shown in FIG. 1A. Tregs are genetically engineered to express the CD137-CD3ζ-CAR which can be determined by expression of LNGFR (FIG. 1B). Antigen-binding of the CD137-CD3ζ-CAR can be determined by incubation with the respective antigen that can be labeled (e.g. fluorescently) as shown for dextran in FIG. 1C.

Example

Example 2

Activation of CAR-Tregs With Different Intracellular Signaling Domains

[0212]CAR-Tregs that are specific for an exogenous antigen (e.g. dextran) can be activated by their respective antigen and activation can be analysed by CD137 expression. CAR-Treg activation after stimulation with bead-bound dextran is shown in FIG. 2A. The CD137-CD3ζ-CAR was more potent in inducing CD137 expression in CAR-Tregs (FIG. 2A). Functionality of other tested CAR-constructs with the same specificity was analysed by phosphorylation of ZAP70. Phosphorylated ZAP70 was detected in CAR-Tregs with e.g. CD28-CD3ζ signaling (FIG. 2B), but only the CD137-CD3ζ-CAR induced Treg activation (FIG. 2A).

Example

Example 3

Expansion of Dextran-Specific CAR-Tregs With Different Intracellular Signaling Domains

[0213]CAR-Tregs that are specific for an exogenous antigen (e.g. dextran) can be expanded in the presence of anti-CD3 / -CD28 (FIG. 3A, C) or their respective antigen, e.g. bead-bound dextran (FIG. 3B, D). Only CAR-Tregs with the CD137-CD3ζ-CAR expanded showing superior functionality of the CD137-CD3ζ-CAR.

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Abstract

The present invention provides a regulatory T (Treg) cell expressing an antigen chimeric receptor (CAR) comprising a) at least one antigen binding domain, b) a transmembrane domain, and c) a cytoplasmic signaling domain comprising at least one primary cytoplasmic signaling domain and at least the co-stimulatory signaling domain of CD137, wherein said antigen binding domain specifically binds an antigen that is expressed on the surface of a target cell or a tag of a tagged polypeptide that binds to an antigen expressed on the surface of a target cell or a soluble antigen. Compositions comprising said Treg cells, and methods of enrichment and analysis of activated Tregs that express said CAR are also disclosed.

Description

FIELD OF THE INVENTION[0001]The present invention relates to the field of chimeric antigen receptors expressed in immune cells, in particular expressed in regulatory T cells.BACKGROUND OF THE INVENTION[0002]Conventional T cells (Tcon) can be manipulated to improve recognition and destruction of pathogens or tumors. Regulatory T cells (Tregs) comprise a subset of T cells with immunosuppressive function. Treg can be manipulated to prevent or treat autoimmunity, transplant rejection, allergy and chronic inflammatory diseases.[0003]Chimeric antigen receptors (CARs) emerge as promising alternative for the generation of antigen-specific regulatory T cells (Tregs). Other than TCRs, CARs are artificial receptors that contain an antibody-type specificity that can bind surface antigens independent of MHC. The specific recognition of particular antigens by CAR-T cells is mediated by antibody-derived single chain variable fragments (scFv) with an extracellular spacer domain that are coupled via...

Claims

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Application Information

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IPC IPC(8): A61K35/17C07K14/705C07K14/725C07K16/44C12N5/0783
CPCC12N5/0637C07K16/44A61K2035/122C07K2319/03C07K14/7051A61K2039/5158A61K35/17C07K14/70517C07K14/70578A61K2039/5156C07K2319/30C07K14/70521C07K2319/02C07K2319/33C07K2317/622A61K39/001A61K39/0008C07K14/705A61P37/08A61P29/00C12N2510/00
Inventor NOWAK, ANNALOCK, DOMINIKKAISER, ANDREWSCHEFFOLD, ALEXANDER
Owner MILTENYI BIOTEC B V & CO KG
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